This project will test the hypothesis that hyperthermia induces specific cellular and/or nuclear changes that sensitize cells to ionizing radiation. A corollary of this hypothesis is that chemical or physical agents that induce the same changes as heat will also cause cells to become radiosensitive. Establishing this hypothesis and its corollary provide the basis of this project: the identification and development of pharmacological agents that induce radiation sensitization utilizing the same molecular pathways as heat shock and can be used to enhance the radiosensitizing effects of moderate hyperthermia. Aim 1: Test the hypothesis that radiation sensitivity can be increased by the aggregation of proteins in the nucleus. To accomplish this aim, we will use cells stably transfected with vectors that conditionally express GFPfusion proteins engineered to spontaneously aggregate in the nucleus or in the cytosol. Aim 2: Identify pharmacological agents that enhance hyperthermic radiosensitization. The NSAID indomethacin is our starting compound. Previous work has established that indomethacin can act a radiosensitizer and enhancer of thermal radiosensitization. Other, independent work, has shown that indomethacin lowers the temperature necessary for the heat induced protein aggregation and lowers the temperature necessary for Hsf-1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves, insufficient for activation. Aim 3) Determine the structural features of inflole analogs that confer thermal radiosensitization. Indomethacin analogs will be synthesized and subjected to a structure-function analysis in order to maximize enhancer activity and minimize toxicity. Analog design will be based on the molecular determinates identified in Aims 1 and 2. That is, we will identify and design analogs with structural features optimized to cause protein aggregation, DNA sensor function inhibition and activation ofNADPH oxidase activity. Aim 4: Determine if the indomethacin analogs identified in Aim 3 enhance thermal radiosensitization in vivo. Two human xenograph tumor models (NSY and HT-29 will be used. Testing will be performed by the in vivo procedures core. The criteria for analog selection will be based on the results obtained from the in vitro performed in Aim 3. Analogs that are the most efficacious in terms of enhancer activity, the least toxic, and exhibit minimal inhibition of cyclooxygenase -1 and -2 will be tested in vivo.